Mitochondria play a pivotal role in cell survival and tissue development by virtue of their role in energy metabolism, regulation of cellular Ca2+ homeostasis and apoptosis. Given this multifactorial role, regulation of cellular Ca2+, metabolism, and bioenergetics function as an integrated system since energy conservation is used to drive each process. Mitochondrial energy conservation (ATP production) requires the respiration-driven formation of a proton electrochemical potential difference (ΔμH) across the inner mitochondrial membrane (IMM), which is created by proton pumping by the respiratory complexes. Maintenance of the gradient demands a low permeability of the IMM to protons, charged species and solutes, whose fluxes are tightly controlled by specific carrier systems that are powered by the two components of the ΔμH, i.e. the membrane potential difference (Δψm) and the ΔpH. Yet, mitochondria in vitro can easily undergo an IMM permeability increase to solutes with molecular masses of about 1,500 Da or lower. This permeability change, called the permeability transition (PT), is regulated by the opening of a membrane pore, the mitochondrial permeability transition pore (MPTP). Long-lasting MPTP opening results in outer mitochondrial membrane (OMM) rupture and cytochrome c release, with ensuing dramatic consequences on mitochondrial function (e.g., collapse of ΔμH, depletion of pyridine nucleotides) that lead to respiratory inhibition. This process has long been studied then, as a target for mitochondrial dysfunction in vivo, particularly in the context of specific human pathological events like ischemia-reperfusion injury and neurodegeneration. The MPTP has also drawn attention as a mediator of programmed cell death (apoptosis) and target of the action of BCL2 family members through the release of cytochrome c (Bernardi, P., Mitochondrial transport of cations: channels, exchangers, and permeability transition. Physiol Rev, 1999. 79(4): p. 1127–55; Nicholls, D. G. and S. L. Budd, Mitochondria and neuronal survival. Physiol Rev, 2000. 80(1): p. 315–60; Bernardi, P., et al., Mitochondria and cell death. Mechanistic aspects and methodological issues. Eur J Biochem, 1999. 264(3): p. 687–701; Bernardi, P., et al., A mitochondrial perspective on cell death. Trends Biochem Sci, 2001. 26(2): p. 112–7).
It is currently agreed that mitochondria play an important role in controlling life and death of cells (apoptosis; Kroemer G & Reed J C, Mitochondrial control of cell death. Nat Med. 2000, 6(5): 513–9). It appears both that an increasing number of molecules involved in the transduction of the signal and also many metabolites and certain viral effectors act on mitochondria and influence the permeabilisation of mitochondrial membranes. Cytoprotective molecules may be used, thanks to their ability to stabilize mitochondrial membranes, in the treatment of illnesses where there is excessive apoptosis (neurodegenerative diseases, ischemia, AIDS, fulminant hepatitis, etc.).
A change in mitochondrial membrane permeability is a key event of apoptotic cell death associated with the release of caspase activators and caspase-independent death effectors from the intermembrane space, dissipation of the inner transmembrane potential, as well as a perturbation of oxidative phosphorylation (Kroemer G & Reed J C, Mitochondrial control of cell death. Nat Med. 2000, 6(5):513–9; Vander Heiden M G & Thompson C B, Bcl-2 proteins: regulators of apoptosis or of mitochondrial homeostasis?, Nat Cell Biol. (1999) 1(8):E209–16; Wallace D C, Mitochondrial diseases in man and mouse. Science (1999); 283 (5407), 1482–8). Pro-and anti-apoptotic members of the Bcl-2 family regulate inner and outer mitochondrial membrane permeability through interactions with the adenine nucleotide translocase (ANT; in the inner membrane), the voltage-dependent anion channel (VDAC; in the outer membrane), and/or through autonomous channel-forming activities (Kroemer G & Reed J C, 2000; Marzo I, Brenner C, Zamzami N, Jurgensmeier J M, Susin S A, Vieira H L, Prevost M C, Xie Z, Matsuyama S, Reed J C, Kroemer G., Bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis. Science, (1998), 281(5385): 2027–31; Shimizu S., Narita M., Tsujimoto Y., Nature (1999), 399, 483–487; Vander Heiden & Thompson, 1999). ANT and VDAC are believed to be major components of the mitochondrial permeability transition pore (MPTP) complex, a polyprotein structure organized at sites at which the two mitochondrial membranes are in close vicinity (Crompton M., Biochem J (1999), 341, 233–249).
The mitochondrial permeability transition pore is a polyprotein complex formed in the contact site between the inner and the outer mitochondrial membranes that participate in the regulation of mitochondrial membrane permeability. It is composed of a set of proteins including mitochondrion-associated hexokinase (HK), porin (voltage-dependent anion channel or VDAC), adenine nucleotide translocation (ANT), peripheral benzodiazepine receptor (PBR), creatine kinase (CK), and cyclophilin D, as well as Bcl-2 family members. In physiological conditions, MPTP controls the mitochondrial calcium homeostasis via the regulation of its conductance by the mitochondrial pH, the mitochondrial membrane potential ΔΨm, NAD/NAD(P)H redox equilibrium and matrix protein thiol oxidation (Shimizu S., Narita M., Tsujimoto Y., Nature (1999), 399, 483–487; Crompton M., Biochem J 341,233–249 (1999); Ichas F., Jouaille L., Mazat J., Cell (1997), 89, 1145–53). MPTP has been implicated in many examples of apoptosis due to its capacity to integrate multiple pro-apoptotic signal transduction pathways and due to its control by proteins from Bcl-2/Bax family. The Bcl-2 family comprises death inhibitory (Bcl-2-like) and death inducing (Bax-like) members which respectively prevent or facilitate MPTP opening. Bax and Bcl-2 reportedly interact with VDAC and ANT within MPTP.
Apoptosis and related forms of controlled cell death are involved in a great number of illnesses. Excess or insufficiency of cell death processes are involved in auto-immune and neurodegenerative diseases, cancers, ischemia, and pathological infections or diseases such as viral and bacterial infections. In the area of neurodegenerative diseases, a great many observations suggest close links with mitochondrial control of apoptosis (see Kroemer G & Reed J C, Mitochondrial control of cell death. Nat Med. (2000), 6(5): 513–9). The neurotoxin-methyl-4-phenylpyridinium induces mitochondrial permeability transition and the exit of cytochrome c (Cassarino D S, Parks J K, Parker W D Jr, Bennett J P Jr. The parkinsonian neurotoxin MPP+ opens the mitochondrial permeability transition pore and releases cytochrome c in isolated mitochondria via an oxidative mechanism. Biochim Biophys Acta 1999;1453, 49–62).
Poisoning by mitochondrial toxins such as nitro-propionic acid or rotenone provokes in primates and rodents a Huntington-disease type of illness (Brouillet E, Hantraye P, Ferrante R J, Dolan R, Leroy-Willig A, Kowall N W, Beal M F., Chronic mitochondrial energy impairment produces selective striatal degeneration and abnormal choreiform movements in primates. Proc Natl Acad Sci USA. 1995 Jul 18;92(15):7105–9; Betarbet R, Sherer T B, MacKenzie G, Garcia-Osuna M, Panov A V, Greenamyre J T. Chronic systemic pesticide exposure reproduces features of Parkinson's disease Nat Neurosci. 2000, 1301–6).
In physiological conditions, ANT is a specific antiporter for ADP and ATP. However, ANT can also form a lethal pore upon interaction with different pro-apoptotic agents, including Ca2+, atractyloside, HIV-Vpr-derived peptides and pro-oxidants. Mitochondrial membrane permeabilization may also be regulated by the non-specific VDAC pore modulated by Bcl-2/Bax-like proteins in the outer membrane and/or by changes in the metabolic ATP/ADP gradient between the mitochondrial matrix and the cytoplasm.
Although the relevance of the MPTP has gained wide recognition for its role in necrotic and apoptotic cell death, much of the information on its molecular identity still relies on indirect evidence. Also, lack of specific high-affinity probes for its components has hindered progress in the field.
More particularly, there exists a need in the art for methods and reagents for investigating and modulating mitochondrial permeabilization and apoptosis.